CN116426632A - Molecular marker and kit for detecting sudden cardiac death by LTBP4 gene polymorphism site - Google Patents
Molecular marker and kit for detecting sudden cardiac death by LTBP4 gene polymorphism site Download PDFInfo
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Abstract
The invention relates to a molecular marker and a kit for detecting sudden cardiac death by using an LTBP4 gene polymorphism site, wherein the molecular marker is an rs34005443 site of an LTBP4 gene, a base CTGT is inserted or deleted in the site, and the insertion type indicates that the risk of sudden cardiac death is high. The invention provides a polymorphic molecular marker related to sudden cardiac death through researching the correlation between the indel polymorphism of the rs34005443 locus of the LTBP4 gene and the sudden cardiac death phenotype, and further provides a sudden cardiac death detection kit, so that the susceptibility of an individual to sudden cardiac death is predicted through detecting and analyzing the rs34005443 locus gene carrying type on the LTBP4 gene of the individual.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a molecular marker and a kit for detecting sudden cardiac death by using LTBP4 gene polymorphic loci.
Background
Sudden cardiac death (sudden cardiac death, SCD) refers to sudden unexpected death caused by cardiac causes, manifested by sudden loss of consciousness and circulatory and respiratory arrest occurring in a short period of time. "short term" is defined as less than 1 hour with witness and 24 hours without witness. Epidemiology shows that 40.7 people of every 10 thousands of people in China die from SCD, and the number of SCD cases is quite large in consideration of the huge population base in China. Numerous studies have previously shown that the cause of death of SCD is mostly coronary atherosclerotic heart disease or fatal cardiac arrhythmias. The former is mostly seen in the elderly population, while the latter is mostly seen in the young population. In view of post mortem autopsy and histological examination, it is difficult to find an accurate cause of death, and in order to accurately diagnose SCD, it is urgent to reveal the molecular mechanism by which SCD occurs and find genetic markers of SCD.
In the past, research in the field of SCD diagnostics has focused mainly on analysis of the association of mutations in the coding region of important protein-encoding genes with sudden death, however, the number of mutations involved is limited, and the extremely low gene frequency of the general population makes it difficult to fully interpret the relatively high incidence of sudden death. With the development of whole genome association studies and second generation sequencing technologies, more and more genetic markers are found to be involved in the regulation of SCD. The identification of the SCD genetic markers can further understand the potential mechanism, optimize the risk stratification of the SCD, and provide powerful theoretical basis for molecular diagnosis and prevention. Considering that the indel polymorphism plays an important role in regulating and controlling gene expression, the indel genetic polymorphism in the region is seen, and the correlation between the indel genetic polymorphism in the related genes and the susceptibility of SCD is analyzed through case contrast research, so that the possibility is provided for establishing a gene polymorphism detection system for SCD risk assessment.
The LTBP4 (latent transforming growth factor-beta binding protein 4) gene encodes a potential transforming growth factor beta (TGF-beta) binding protein 4, which plays a key role in the regulation of TGF-beta signaling. TGF-beta signaling is involved in the development and maintenance of cardiovascular tissues, including blood vessels and heart valves, and plays a role in various cardiovascular pathologies by regulating the expression of related genes.
In the prior art, no research report on the correlation between rs34005443 indel polymorphism and SCD exists, and no relevant report on the prediction of the susceptibility of SCD by detecting the indel polymorphic site of 3' UTR of LTBP4 gene exists.
Disclosure of Invention
In order to solve the technical problems, the invention provides a sudden cardiac death detection kit for detecting the risk of sudden cardiac death of an individual by verifying that a four-base (CTGT) indel polymorphism (rs 34005443) in an intron region of an LTBP4 gene is obviously associated with the risk of suffering from SCD for the first time.
The first object of the invention is to provide an indel polymorphism molecular marker for detecting the risk of sudden cardiac death, wherein the molecular marker is the rs34005443 locus of the LTBP4 gene.
Further, the rs34005443 site has a 4 base indel polymorphism, specifically, an indel or deleted base CTGT.
Furthermore, the insertion type indicates a high risk of sudden cardiac death and the deletion type indicates a low risk of sudden cardiac death.
The second object of the invention is to provide an application of the LTBP4 gene indel polymorphism site in preparing a sudden cardiac death detection kit, wherein the kit is used for detecting the genotype of the LTBP4 gene rs34005443 site.
Further, the sudden cardiac death detection kit comprises a primer pair for amplifying the rs34005443 locus of the LTBP4 gene.
Further, the primer pair for amplifying the locus rs34005443 of the LTBP4 gene comprises a sense primer and an antisense primer, the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, the nucleotide sequence of the antisense primer is shown as SEQ ID NO.2, and specific sequence information is as follows:
SEQ ID NO.1：5′-CATGCTCCTGGCTCGAC-3′，
SEQ ID NO.2：5′-TGCCTTTAACCACCTAGCCA-3′。
further, for ease of identification, the primer pair is provided with an identification label, such as a fluorescent label.
The primer pair is designed for the rs34005443 indel site on the LTBP4 gene, can specifically amplify a DNA fragment containing the rs34005443 indel site on the LTBP4 gene, marks fluorescent dye at the 5' end of the oligonucleotide primer by a fluorescent marking technology, and one chain of a PCR amplified product carries the fluorescent dye of the marked primer. It will be appreciated by those skilled in the art that specific primer pairs can be synthesized using conventional synthetic techniques, and that the primers of the present invention are not limited to the pair of primers SEQ ID NO.1 and SEQ ID NO. 2.
Further, the Tm value of the sense primer was 57℃and the Tm value of the antisense primer was 59 ℃.
Further, the sudden cardiac death detection kit also comprises components for PCR amplification and capillary electrophoresis.
Further, the components for PCR amplification and capillary electrophoresis include Taq DNA polymerase, dNTP mixture, mgCl 2 Solution, PCR reaction buffer and deionized water. The amplified products of the PCR are analyzed for the genotype of the site by capillary electrophoresis separation techniques. The components of the assembly for PCR amplification and capillary electrophoresis and the preparation method thereof are all in the prior art.
In one embodiment of the invention, the components and amounts of the kit include 50. Mu.M specific primer pairs, 2.5U/. Mu.L Taq DNA polymerase; 2.5mM dNTP mix; 25mM MgCl 2 A solution; 10 XPCR reaction buffer; deionized water.
Further, the preservation temperature of the kit is-20 ℃.
The principle of the invention is as follows: the inventor discovers through molecular biological research that different isogenotypes of one insertion deletion polymorphism rs34005443 in an intron region of the LTBP4 gene can encode various proteins by influencing variable shearing of mRNA of the LTBP4 gene and possibly interact with promoters of adjacent genes as regulatory elements to remotely regulate the transcriptional activity of the adjacent genes; furthermore, it was found from SCD case-control studies in a population of a certain scale that the insertion allele of the insertion deletion polymorphism was positively correlated with the occurrence of SCD, and that this correlation was still present even after adjustment of age, sex, etc. This indel polymorphism is therefore considered useful for assessing the susceptibility of an individual to SCD.
The third object of the invention is to provide an application of a primer for amplifying the rs34005443 locus of the LTBP4 gene in preparing a sudden cardiac death detection kit, wherein the primer can identify the genotype of the rs34005443 locus of the LTBP4 gene.
Further, the primer for amplifying the locus rs34005443 of the LTBP4 gene comprises a sense primer and an antisense primer, the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, the nucleotide sequence of the antisense primer is shown as SEQ ID NO.2, and specific sequence information is as follows:
SEQ ID NO.1：5′-CATGCTCCTGGCTCGAC-3′，
SEQ ID NO.2：5′-TGCCTTTAACCACCTAGCCA-3′。
by means of the scheme, the invention has at least the following advantages:
the specific primer pair contained in the kit is designed aiming at an rs34005443 indel site on the LTBP4 gene, DNA fragments containing the site can be specifically amplified, different genotypes can be identified by detecting the mobility of fragments with different lengths in capillary electrophoresis, and according to the research of case contrast, the rs34005443 indel site on the LTBP4 gene of the detected DNA can be considered to carry an indel allele and is of SCD susceptibility type. Therefore, the technology can play a role in predicting the susceptibility of the individual SCD by detecting the genotype of the rs34005443 indel site on the individual LTBP4 gene.
The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.
FIG. 1 shows the gene frequency differences and the risk of disease OR values at this site for the control and SCD case groups;
FIG. 2 is a diagram showing gene sequencing and SDS-PAGE gel electrophoresis typing.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example 1 use of a detection kit:
step 1: extraction of DNA template
Genomic DNA of peripheral blood was extracted using a blood genomic DNA extraction system (non-centrifugal column).
Step 2: PCR reaction-replication of the fragment of interest
A PCR detection kit was used that can detect SCD susceptibility, containing the following primers:
SEQ ID No.1:5'-CATGCTCCTGGCTCGAC-3', tm is 57 ℃;
SEQ ID No.2:5'-TGCCTTTAACCACCTAGCCA-3', tm is 59 ℃;
the primer pair can specifically amplify fragments containing the rs34005443 insertion deletion site polymorphism in the LTBP4 gene.
The total volume of the PCR reaction system was 10ul, including: 1 μl of DNA template, 50 μl of each of the 50 μM specific primer pairs, 0.04 μl each, 2.5U/μl Taq DNA polymerase 0.08 μl each; 0.2. Mu.l of 2.5mM dNTP mix; 25mM MgCl 2 0.6. Mu.l of solution; 1 μl of 10 XPCR reaction buffer; the deionized water is replenished; the reaction was performed on a Eppendorf Mastercycler nexus PCR amplification apparatus under the following conditions: 94 ℃ for 3min; a further 30 PCR cycles were performed: 94 ℃ for 30s,59 ℃ for 30s, and 72 ℃ for 1min; finally, the temperature is 72 ℃ for 5min.
Step 3: indel genotyping
And (3) performing capillary electrophoresis separation on the amplified product by using an ABI 3500 gene sequencer to obtain the genotype of the detected individual, and providing explanation by a professional.
The experimental effect of the invention:
the differences in gene frequency and risk of disease OR values at this site for the control and SCD case groups are shown in figure 1.
As shown in fig. 1, in the co-dominant model, within the 95% confidence interval, genotype is 1.815 times the risk of developing SCD for an insert/insert type individual than for a deletion/deletion type individual, and genotype is 1.950 times the risk of developing SCD for an insert/deletion type individual than for a deletion/deletion type individual; in the dominant model, individuals with genotype insertion/insertion or insertion/deletion are at 1.925 times the risk of developing SCD than individuals with deletion/deletion; in the additive model, individuals carrying the insertion allele are at 1.434 times more risk of developing SCD than individuals carrying the deletion allele.
The gene sequencing diagram and SDS-PAGE gel electrophoresis typing diagram are shown in FIG. 2.
Wherein, FIG. 2A is an example of a template strand sequencing result, corresponding to a four base indel at rs34005443 for the coding strand at the underline; FIG. 2B shows the result of electrophoresis of 12 DNA samples from different individuals using the PCR amplification system of the present invention, wherein 1, 2, 7 are inserted homozygotes, 4, 6, 10 are deleted homozygotes, and the rest are heterozygotes. The left and right are DNA markers.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. An indel polymorphism molecular marker for detecting risk of sudden cardiac death onset, characterized in that: the insertion deletion polymorphism molecular marker is an rs34005443 locus of the LTBP4 gene, and the locus is inserted into or deleted from a base CTGT.
2. The indel polymorphic molecular marker of claim 1, wherein: insertion indicates a high risk of sudden cardiac death.
The application of LTBP4 gene indel polymorphism site in preparing sudden cardiac death detection kit is characterized in that: the sudden cardiac death detection kit is used for detecting the genotype of the rs34005443 locus of the LTBP4 gene.
4. A use according to claim 3, characterized in that: the sudden cardiac death detection kit comprises a primer for amplifying the rs34005443 locus of the LTBP4 gene.
5. The use according to claim 4, characterized in that: the primer for amplifying the locus rs34005443 of the LTBP4 gene comprises a sense primer and an antisense primer, wherein the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense primer is shown as SEQ ID NO. 2.
6. The use according to claim 4, characterized in that: the primer for amplifying the rs34005443 locus of the LTBP4 gene is provided with an identification mark.
7. A use according to claim 3, characterized in that: the sudden cardiac death detection kit also comprises components for PCR amplification and capillary electrophoresis.
8. Application of primer for amplifying LTBP4 gene rs34005443 locus in preparation of sudden cardiac death detection kit.
9. The use according to claim 8, characterized in that: the primer for amplifying the locus rs34005443 of the LTBP4 gene comprises a sense primer and an antisense primer, wherein the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense primer is shown as SEQ ID NO. 2.
10. A kit for detecting sudden cardiac death based on LTBP4 gene indel polymorphism is characterized in that: the sudden cardiac death detection kit is used for detecting the genotype of the rs34005443 locus of the LTBP4 gene and comprises a primer for amplifying the rs34005443 locus of the LTBP4 gene.
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